This proposal describes a logical "front end" cloning and template generation system that can be readily automated (Figure 1) 1) The first step is the development of a cloning vector (in this case a cosmid called pCOSEQ) having a number of unique features aimed at optimizing mega-mapping and mega-sequencing. 2) The second step is to construct a chromosome specific human genomic library in this vector. 3) The third step is to use the unique features of the cloning vector to physically order the chromosome specific clones (this proposal covers the development of the method to order the clones and the ordering of a small number of clones ; the labor involved in ordering a chromosome specific library will not be in the budget of this proposal). 4) The fourth step is to pick order cosmid clones and rapidly generate a set of clones containing ordered nested deletions using a methodology amenable to automation. 5) The fifth step is to generate sequence information directly from the cosmid clones, without subcloning, in a manner that is amenable to automation. The successful completion of this project should lay much of the groundwork necessary for an economical approach to physically mapping and sequencing the human genome. Let it be clearly stated that Stratagene is undertaking this program as a basic research project and would make all resulting materials widely available for non-commercial research purposes.